Method for the complete chemical synthesis and assembly of genes and genomes

ABSTRACT

The present invention relates generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms. Thus, the present invention outlines a novel approach to utilizing the results of genomic sequence information by computer directed gene synthesis based on computing on the human genome database. Specifically, the present invention contemplates and describes the chemical synthesis and resynthesis of genes defined by the genome sequence in a host vector and transfer and expression of these sequences into suitable hosts.

This application claims the benefit of No. 60/059,017, filed Sep. 16, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms.

2. Description of Related Art

Present research and commercial applications in molecular biology are based upon recombinant DNA developed in the 1970's. A critical facet of recombinant DNA is molecular cloning in plasmids, covered under seminal patent of Cohen and Boyer (U.S. Pat. No. 4,740,470 “Biologically functional molecular chimeras”). This patent teaches a method for the “cutting and splicing” of DNA molecules based upon restriction endonucleases, the introduction of these “recombinant” molecules into host cells, and their replication in the bacterial hosts. This technique is the basis of all molecular cloning for research and commercial purposes carried out for the past 20 years and the basis of the field of molecular biology and genetics.

Recombinant DNA technology is a powerfull technology, but is limited in utility to modifications of existing DNA sequences which are modified through 1) restriction enzyme cleavage sites, 2) PAC primers for amplification, 3) site-specific mutagenesis, and other techniques. The creation of an entirely new molecule, or the substantial modification of existing molecules, is extremely time consuming, expensive, requires complex and multiple steps, and in some cases is impossible. Recombinant DNA technology does not permit the creation of entirely artificial molecules, genes, genomes or organisms, but only modifications of naturally-occurring organisms.

Current biotechnology for industrial production, for drug design and development, for potential applications of vaccine development and genetic therapy, and for agricultural and environmental use of recombinant DNA, depends on naturally-occurring organisms and DNA molecules. To create or engineer new or novel functions, or to modify organisms for specialized use (such as producing a human hormone), requires substantially complex, time consuming and difficult manipulations of naturally-occurring DNA molecules. In some cases, changes to naturally-occurring DNA are so complex that they are not possible in practice. Thus, there is a need for technology that allows the creation of novel DNA molecules in a single step without requiring the use of any existing recombinant or naturally-occurring DNA.

SUMMARY OF THE INVENTION

The present invention addresses the limitations in present recombinant nucleic acid manipulations by providing a fast, efficient means for generating practically any nucleic acid sequence, including entire genes, chromosomal segments, chromosomes and genomes. Because this approach is based on an completely synthetic approach, there are no limitations, such as the availability of existing nucleic acids, to hinder the construction of even very large segments of nucleic acid.

Thus, in a first embodiment, there is provided a method for the construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of said DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing said first and said second set of oligonucleotides, and (iii) treating said annealed oligonucleotides with a ligating enzyme. Optional steps provide for the synthesis of the oligonucleotide sets and the transformation of host cells with the resulting DNA segment.

In particular embodiments, the DNA segment is 100, 200, 300, 40,, 800, 100, 1500, 200, 4000, 8000, 10000, 12000, 18,000, 20000, 40,000, 80,000; 100,000, 10⁶, 10⁷, 10⁸, 10⁹ or more base pairs in length. Indeed, it is contemplated that the methods of the present invention will be able to create entire artificial genomes of lengths comparable to known bacterial, yeast, viral, mammalian, amphibian, reptilian, avian genomes. In more particular embodiments, the DNA segment is a gene encoding a protein of interest. The DNA segment further may include non-coding elements such as origins of replication, telomeres, promoters, enhancers, transcription and translation start and stop signals, introns, exon splice sites, chromatin scaffold components and other regulatory sequences. The DNA segment may comprises multiple genes, chromosomal segments, chromosomes and even entire genomes. The DNA segments may be derived from prokaryotic or eukaryotic sequences including bacterial, yeast, viral, mammalian, amphibian, reptilian, avian, plants, archebacteria and other DNA containing living organisms.

The oligonucleotide sets preferably are comprised oligonucleotides of between about 15 and 100 bases and more preferably between about 20 and 50 bases. Specific lengths include, but are not limited to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64.65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100. Depending on the size, the overlap between the oligonucleotides of the two sets may be designed to be between 5 and 75 bases per oligonucleotide pair.

The oligonucleotides preferably are treated with polynucleotide kinase, for example, T4 polynucleotide kinase. The kinasing can be performed prior to mixing of the oligonucleotides set or after, but before annealing. After annealing, the oligonucleotides are treated with an enzyme having a ligating function. For example, a DNA ligase typically will be employed for this function. However, topoisomerase, which does not require 5′ phosphorylation, is rapid and operates at room temperature, and may be used instead of ligase.

In a second embodiment, there is provided a method for construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of said DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing pairs of complementary oligonucleotides to produce a set of first annealed products, wherein each pair comprises an oligonucleotide from each of said first and said second sets of oligonucleotides, (iii) annealing pairs of first annealed products having complementary sequences to produce a set of second annealed products, (iv) repeating the process until all annealed products have been annealed into a single DNA segment, and (v) treating said annealed products with ligating enzyme.

In a third embodiment, there is provided a method for the construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of sand DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing said the 5′ terminal oligonucleotide of said first set of oligonucleotide with the 3′ terminal oligonucleotide of said second set of oligonucleotides, (iii) annealing the next most 5′ terminal oligonucleotide of said first set of oligonucleotides with the product of step (ii), (iv) annealing the next most 3′ terminal oligonucleotide of said second set of oligonucleotides with the product of step (iii), (v) repeating the process until all oligonucleotides of said first and said second sets have been annealed, and (vi) treating said annealed oligonucleotides with ligating enzyme. Optional steps provide for the synthesis of the oligonucleotide sets and the transformation of host cells with the resulting DNA segment. In a preferred embodiment, the 5′ terminal oligonucleotide of the first set is attached to a support, which process may include the additional step of removing the DNA segment from the support. The support may be any support known in the art, for example, a microtiter plate, a filter, polystyrene beads, polystyrene tray, magnetic beads, agarose and the like.

Annealing conditions may be adjusted based on the particular strategy used for annealing, the size and composition of the oligonucleotides, and the extent of overlap between the oligonucleotides of the first and second sets. For example, where all the oligonucleotides are mixed together prior to annealing, heating the mixture to 80° C., followed by slow annealing for between 1 to 12 h is conducted. Thus, annealing may be conducted for about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 h. However, in other embodiments, the annealing time may be as long as 24 h.

With the aid of a computer, the inventor is able to direct synthesis of a vector/gene combination using a high throughput oligonucleotide synthesizer as a set of overlapping component oligonucleotides. The oligonucleotides are assembled using a robotic combinatoric assembly strategy and the assembly ligated using DNA ligase or topoisomerase, followed by transformation into a suitable host strain. In a particular embodiment, this invention generates a set of bacterial strains containing a viable expression vector for all genes in a defined region of the genome. In other embodiments, a yeast or baculovirus expression vector system is also contemplated to allow expression of each gene in a chromosomal region in a eukaryotic host. In yet another embodiment, it the present invention allows one of skill in the art to devise a “designer gene” strategy wherein a gene or genomes or virtually any structure may be readily designed, synthesized and expressed. Thus, eventually the technology described herein may be employed to create entire genomes for introduction into host cells for the creation of entirely artificial designer living organisms.

In specific embodiments, the present invention provides a method for the synthesis of a replication-competent, double-stranded polynucleotide, wherein the polynucleotide comprises an origin of replication, a first coding region and a first regulatory element directing the expression of the first coding region.

Additionally the method may further comprise the step of amplifying the double-stranded polynucleotide. In specific embodiments, the double-stranded polynucleotide comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10×10³, 20×10³, 30×10³, 40×10³, 50×10³, 60×10³, 70×10³, 80×10³, 90×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹ or 1×10¹⁰ base pairs in length. The first regulatory element may be a promoter. In certain embodiments, the double-stranded polynucleotide further comprises a second regulatory element, the second regulatory element being a polyadenylation signal. In yet further embodiments, the double-stranded polynucleotide comprises a plurality of coding regions and a plurality of regulatory elements. Specifically, it is contemplated that the coding regions encode products that comprise a biochemical pathway. In particular embodiments the biochemical pathway is glycolysis. More particularly, it is contemplated that the coding regions encode enzymes selected from the group consisting of hexokinase, phosphohexose isomerase, phosphofructokinase-1, aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase enzymes of the glycolytic pathway.

In other embodiments, the biochemical pathway is lipid synthesis, cofactor synthesis. Particularly contemplated are synthesis of lipoic acid, riboflavin synthesis nucleotide synthesis. the nucleotide may be a purine or a pyrimidine.

In certain other embodiments it is contemplated that the coding regions encode enzymes involved in a cellular process selected from the group consisting of cell division, chaperone, detoxification, peptide secretion, energy metabolism, regulatory function, DNA replication, transcription, RNA processing and tRNA modification. In preferred embodiments, the energy metabolism is oxidative phosphorylation.

It is contemplated that the double-stranded polynucleotide is a DNA or an RNA. In preferred embodiments, the double-stranded polynucleotide may be a chromosome. The double-stranded polynucleotide may be an expression construct. Specifically, the expression construct may be a bacterial expression construct, a mammalian expression construct or a viral expression construct. In particular embodiments, the double-stranded polynucleotide comprises a genome selected from the group consisting of bacterial genome, yeast genome, viral genome, mammalian genome, amphibian genome and avian genome.

In those embodiments in which the genome is a viral genome, the viral genome may be selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.

The present invention further provides a method of producing a viral particle.

Another embodiment provides a method of producing an artificial genome, wherein the chromosome comprises all coding regions and regulatory elements found in a corresponding natural chromosome. In specific embodiments, the corresponding natural chromosome is a human mitochondrial genome. In other embodiments, the corresponding natural chromosome is a chloroplast genome.

Also provided is a method of producing an artificial genetic system, wherein the system comprises all coding regions and regulatory elements found in a corresponding natural biochemical pathway. Such a biochemical pathway will likely possess a group of enzymes that serially metabolize a compound. In particularly preferred embodiments, the biochemical pathway comprises the activities required for glycolysis. In other embodiments, the biochemical pathway comprises the enzymes required for electron transport. In still further embodiments, the biochemical pathway comprises the enzyme activities required for photosynthesis.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Flow diagram of the Jurassic Park paradigm for the reassembly of living organisms.

FIG. 2. Flow diagram of the strategy of synthetic genetics.

FIG. 3. Flow diagram of the strategy for combinatoric assembly of oligonucleotides into complete genes or genomes.

FIG. 4. Design of plasmid synlux4. The sequence of 4800 is annotated with the locations of lux A+B genes, neomycin/kanamycin phosphotransferase and pUC19 sequences.

FIG. 5. List of component oligonucleotides derived from the sequence of Synlux4 in FIG. 4.

FIG. 6. Schema for the combinatoric assembly of synthetic plasmids from component oligonucleotides.

FIG. 7. SynGene program for generating overlapping oligonucleotides sufficient to reassemble the gene or plasmid.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The complete sequence of complex genomes, including the human genome, make large scale functional approaches to genetics possible. The present invention outlines a novel approach to utilizing the results of genomic sequence information by computer directed gene synthesis based on computing on the human genome database. Specifically, the invention describes chemical synthesis and resynthesis of genes for transfer of these genes into a suitable host cells.

The present invention provides methods that can be used to synthesize de novo, DNA segments that encode sets of genes, either naturally occurring genes expressed from natural or artificial promoter constructs or artificial genes derived from synthetic DNA sequences, which encodes elements of biological systems that perform a specified function or attribution of an artificial organism as well as entire genomes. In producing such systems and genomes, the present invention provides the synthesis of a replication-competent, double-stranded polynucleotide, wherein the polynucleotide has an origin of replication, a first coding region and a first regulatory element directing the expression of the first coding region. By replication competent, it is meant that the polynucleotide is capable of directing its own replication. Thus, it is envisioned that the polynucleotide will possess all the cis-acting signals required to facilitate its own synthesis. In this respect, the polynucleotide will be similar to a plasmid or a virus, such that once placed within a cell, it is able to be replicated by a combination of the polynucleotide's and cellular functions.

Thus, using the techniques of the present invention, one of skill in the art can create an artificial genome that is capable of encoding all the activities required for sustaining its own existence. Also contemplated are artificial genetic systems that are capable of encoding enzymes and activities of a particular biochemical pathway. In such a system, it will be desirable to have all the activities present such that the whole biochemical pathway will operate. The co-expression of a set of enzymes required for a particular pathway constitutes a complete genetic or biological system. For example, the co-expression of the enzymes involved in glycolysis constitutes a complete genetic system for the production of energy in the form of ATP from glucose. Such systems for energy production may include groups of enzymes which naturally or artificially serially metabolize a set of compounds.

The types of biochemical pathways would include but are not limited to those for the biosynthesis of cofactors prosthetic groups and carriers (lipoate synthesis, riboflavin synthesis pyridine nucleotide synthesis); the biosynthesis of the cell envelopes (membranes, lipoproteins, porins, surface polysaccharides, lipopolysaccharides, antigens and surface structures); cellular processes including cell division, chaperones, detoxification, protein secretion, central intermediary metabolism (energy production vi phosphorus compounds and other); energy metabolism including aerobic, anaerobic, ATP proton motive force interconversions, electron transport, glycolysis triose phosphate pathway, pyruvate dehydrogenase, sugar metabolism; purine, pyrimidine nucleotide synthesis, including 2′deoxyribonucleotide synthesis, nucleotide and nucleoside interconversion, salvage of nucleoside and nucleotides, sugar-nucleotide biosynthesis and conversion; regulatory functions including transcriptional and translational controls, DNA replication including degradation of DNA, DNA replication, restriction modification, recombination and repair; transcription including degradation of DNA, DNA-dependent RNA polymerase and transcription factors; RNA processing; translation including amino acyl tRNA synthetases, degradation of peptides and glycopeptides, protein modification, ribosome synthesis and modification, tRNA modification; translation factors transport and binding proteins including amino acid, peptide, amine carbohydrate, organic alcohol, organic acid and cation transport; and other systems for the adaptation, specific function or survival of an artificial organism.

A. Definitions

DNA segment—a linear piece of DNA having a double-stranded region and both 5′- and 3′-ends; the segment may be of any length sufficiently long to be created by the hybridization of at least two oligonucleotides have complementary regions.

Oligonucleotides—small DNA segments, single-stranded or double-stranded, comprised of the nucleotide bases A, T, G and C linked through phosphate bonds; oligonucleotides typically range from about 10 to 100 base pairs.

Plus strand—by convention, the single-strand of a double-stranded DNA that starts with the 5′ end to the left as one reads the sequence.

Minus strand—by convention, the single-strand of a double-stranded DNA that starts with the 3′ end to the left as one reads the sequence.

Complementary—where two nucleic acids have at least a portion of their sequences, when read in opposite (5′→3′; 3′→5′) direction, that pair sequential nucleotides in the following fashion: A-T, G-C, T-A, G-C.

Oligonucleotide sets—a plurality of oligonucleotides that, taken together, comprise the sequence of a plus or minus strand of a DNA segment.

Annealed products—two or more oligonucleotides having complementary regions, where they are permitted, under proper conditions, to base pair, thereby producing double stranded regions.

B. The Present Invention

The present invention describes methods for enabling the creation of DNA molecules, genomes and entire artificial living organisms based upon information only, without the requirement for existing genes, DNA molecules or genomes.

The methods of the present invention are diagrammed in FIG. 1 and FIG. 2 and generally involve the following steps. Generally, using simple computer software, comprising sets of gene parts and functional elements it is possible to construct a virtual polynucleotide in the computer. This polynucleotide consists of a string of DNA bases, G, A, T or C, comprising for example an entire artificial genome in a linear string. For transfer of the synthetic gene into for example, bacterial cells the polynucleotide should contain the sequence for a bacterial (such as pBR322) origin of replication. For transfer into eukaryotic cells, it should contain the origin of replication of a mammalian virus, chromosome or subcellular component such as mitochondria.

Following construction, simple computer software is then used to break down the genome sequence into a set of overlapping oligonucleotides of specified length. This results in a set of shorter DNA sequences which overlap to cover the entire genome in overlapping sets. Typically, a gene of 1000 bases pairs would be broken down into 20 100-mers where 10 of these comprise one strand and 10 of these comprise the other strand. They would be selected to overlap on each strand by 25 to 50 base pairs.

This step is followed by direction of chemical synthesis of each of the overlapping set of oligonucleotides using an array type synthesizer and phosphoamidite chemistry resulting in an array of synthesized oligomers. The next step is to balance concentration of each oligomer and pool the oligomers so that a single mixture contains equal concentrations of each. The mixed oligonucleotides are treated with T4 polynucleotide kinase to 5′ phosphorylate the oligonucleotides. The next step is to carry out a “slow” annealing step to co-anneal all of the oligomers into the sequence of the predicted gene or genome. This is done by heating the mixture to 80° C., then allowing it to cool slowly to room temperature over several hours. The mixture of oligonucleotides is then treated with T4 DNA ligase (or alternatively topoisomerase) to join the oligonucleotides. The oligonucleotides are then transferred into competent host cells.

The above technique represents a “combinatorial” assembly strategy where all oligonucleotides are jointly co-annealed by temperature-based slow annealing. A variation on this strategy, which may be more suitable for very long genes or genomes, such as greater than 5,000 base pairs final length, is as follows. Using simple computer software, comprising sets of gene parts and functional elements, a virtual gene or genome is constructed in the computer. This gene or genome would consist of a string of DNA bases, G, A, T or C, comprising the entire genome in a linear string. For transfer of the synthetic gene into bacterial cells, it should contain the sequence for a bacterial (such as pBR322) origin of replication.

The next step is to carry out a ligation chain reaction using a new oligonucleotide addition each step. With this procedure, the first oligonucleotide in the chain is attached to a solid support (such as an agarose bead). The second is added along with DNA ligase, and annealing and ligation reaction carried out, and the beads are washed. The second, overlapping oligonucleotide from the opposite strand is added, annealed and ligation carried out. The third oligonucleotide is added and ligation carried out. This procedure is replicated until all oligonucleotides are added and ligated. This procedure is best carried out for long sequences using an automated device. The DNA sequence is removed from the solid support, a final ligation (is circular) is carried out, and the molecule transferred into host cells.

Alternatively, it is contemplated that if the ligation kinetics allow all the oligonucleotides may be placed in a mixture and ligation be allowed to proceed. In yet another embodiment, a series of smaller polynucleotides may be made by ligating 2, 3, 4, 5, 6, or 7 oligonucleotides into one sequence and adding this to another sequence comprising a similar number of oligonucleotides parts.

The ligase chain reaction (“LCR”), disclosed in EPO No. 320 308, is incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LAC for binding probe pairs to a target sequence. The following sections describe these methods in further detail.

C. Nucleic Acids

The present invention discloses the artificial synthesis of genes. In one embodiment of the present invention, the artificial genes can be transferred into cells to confer a particular function either as discrete units or as part of artificial chromosomes or genome. One will generally prefer to design oligonucleotides having stretches of 15 to 100 nucleotides, 25 to 200 nucleotides or even longer where desired. Such fragments may be readily prepared by, directly synthesizing the fragment by chemical means as described below.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of hybrization selectivity. Typically high selectivity is favored.

For applications requiring high selectivity, one typically will desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50° C. to about 70° C. Such high stringency conditions tolerate little, if any, mismatch between the oligonucleotide and the template or target strand. It generally is appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

For certain applications, for example, by analogy to, substitution of nucleotides by site-directed mutagenesis, it is appreciated that lower stringency conditions may be used. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated depending on the desired results.

In certain embodiments, it will be advantageous to determining the hybridization of ilogonucleotides by employing as a label. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means visible to the human eye or spectrophotometrically,to identify whether specific hybridization with complementary oligonucleotidehas occured.

In embodiments involving a solid phase, for example the first oligonucleotide is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with the complementary oligonucleotides under desired conditions. The selected conditions will also depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface to remove non-specifically bound oligonucleotides, the hybridization may be detected, or even quantified, by means of the label.

For applications in which the nucleic acid segments of the present invention are incorporated into vectors, such as plasmids, cosmids or viruses, these segments may be combined with other DNA sequences, such as promoters, polyadenylation signals, restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

DNA segments encoding a specific gene may be introduced into recombinant host cells and employed for expressing a specific structural or regulatory protein. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected genes may be employed. Upstream regions containing regulatory regions such as promoter regions may be isolated and subsequently employed for expression of the selected gene.

The nucleic acids employed may encode antisense constructs that hybridize, under intracellular conditions, to a nucleic acid of interest. The term “antisense construct” is intended to refer to nucleic acids, preferably oligonucleotides, that are complementary to the base sequences of a target DNA. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target nucleic acid and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene.

Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., a ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).

According to the present invention, DNA segments of a variety of sizes will be produced. These DNA segments will, by definition, be linear molecules. As such, they typically will be modified before further use. These modifications include, in one embodiment, the restriction of the segments to produce one or more “sticky ends” compatible with complementary ends of other molecules, including those in vectors capable of supporting the replication of the DNA segment. This manipulation facilitates “cloning” of the segments.

Typically, cloning involves the use of restriction endonucleases, which cleave at particular sites within DNA strands, to prepare a DNA segment for transfer into a cloning vehicle. Ligation of the compatible ends (which include blunt ends) using a DNA ligase completes the reaction. Depending on the situation, the cloning vehicle may comprises a relatively small portion of DNA, compared to the insert. Alternatively, the cloning vehicle may be extremely complex and include a variety of features that will affect the replication and function of the DNA segment. In certain embodiments, a rare cutter site may be introduced into the end of the polynucleotide sequence.

Cloning vehicles include plasmids such as the pUC series, Bluescript™ vectors and a variety of other vehicles with multipurpose cloning sites, selectable markers and origins of replication. Because of the nature of the present invention, the cloning vehicles may include such complex molecules as phagemids and cosmids, which hold relatively large pieces of DNA. In addition, the generation of artificial chromosomes, and even genomes.

Following cloning into a suitable vector, the construct then is transferred into a compatible host cell. A variety of different gene transfer techniques are described elsewhere in this document. Culture of the host cells for the intended purpose (amplification, expression, subcloning) follows.

Throughout this application, the term “expression construct” is meant to include a particular kind of cloning vehicle containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs.

In preferred embodiments, the nucleic acid is under transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. Preferred promoters include those derived from HSV. Another preferred embodiment is the tetracycline controlled promoter.

In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose. It is envisioned that any elements/promoters may be employed in the context of the present invention. Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Enhancer/promoter elements contemplated for use with the present invention include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light, Chain T-Cell Receptor, HLA DQ α and DQ β, β-Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRα, β-Actin, Muscle Creatine Kinase, Prealbumin (Transthyretin), Elastase I, Metallothionein, Collagenase, Albumin Gene, α-Fetoprotein, τ-Globin, β-Globin, e-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), α1-Antitrypsin, H2B (TH2B) Histone, Mouse or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor, Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus, Gibbon Ape Leukemia Virus. Inducible promoter elements and, their associated inducers are listed in Table 2 below. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

TABLE 2 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV (mouse mammary tumor Glucocorticoids virus) β-Interferon poly(rI)X poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ Collagenase Phorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid Hormone Gene

Use of the baculovirus system will involve high level expression from the powerful polyhedron promoter.

One will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements (Bittner et al., 1987).

In certain embodiments, it may be desirable to include specialized regions known as telomeres at the end of a genome sequence. Telomeres are repeated sequences found at chromosome ends and it has long been known that chromosomes with truncated ends are unstable, tend to fuse with other chromosomes and are otherwise lost during cell division. Some data suggest that telomeres interaction the nucleoprotein complex and the nuclear matrix. One putative role for telomeres includes stabilizing chromosomes and shielding the ends from degradative enzyme.

Another possible role for telomeres is in replication. According to present doctrine, replication of DNA requires starts from short RNA primers annealed to the 3′-end of the template. The result of this mechanism is an “end replication problem” in which the region corresponding to the RNA primer is not replicated. Over many cell divisions, this will result in he progressive truncation of the chromosome. It is thought that telomeres may provide a buffer against this effect, at least until they are themselves eliminated by this effect. A further structure to be included in DNA segments is a centromere.

In certain embodiments of the invention, the delivery of a nucleic acid in a cell may be identified in vitro or in vivo by including a marker in the expression construct. The marker would result in an identifiable change to the transfected cell permitting easy identification of expression.

A number of selection systems may be used, including, but not limited, to the herpes simplex virus thymidine kinase (Wigler et al., 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962) and adenine phosphoribosyltransferase genes (Lowy et al., 1980), in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980; O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid (Mulligan et al., 1981); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981); and hygro, which confers resistance to hygromycin.

Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

In certain embodiments of the invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.

D. Encoded Proteins

In this application, the inventors use genetic information for creative or synthetic purposes. The complete genome sequence will give a catalog of all genes necessary for the survival, reproduction, evolution and speciation of an organisms and, given suitable high tech tools, the genomic information may be modified or even created from “scratch” in order to synthesize life. Thus it is contemplated that a combination of suitable energy generation genes, regulatory genes, and other functional genes could be constructed which would be sufficient to render an artificial organism with the basic functionalities to enable independent survival.

To meet this goal, the present invention utilizes known cDNA sequences for any given gene to express proteins in an artificial organism. Any protein so expressed in this invention may be modified for particular purposes according to methods well known to those of skill in the art. For example, particular peptide residues may be derivatized or chemically modified in order to alter the immune response or to permit coupling of the peptide to other agents. It also is possible to change particular amino acids within the peptides without disturbing the overall structure or antigenicity of the peptide. Such changes are therefore termed “conservative” changes and tend to rely on the hydrophilicity or polarity of the residue. The size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative.

Once the entire coding sequence of a gene has been determined, the gene can be inserted into an appropriate expression system. The gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used to vaccinate animals to generate antisera with which further studies may be conducted.

Examples of expression systems known to the skilled practitioner in the art include bacteria such as E. coli, yeast such as Saccharomyces cerevisia and Pichia pastoris, baculovirus, and mammalian expression systems such as in COS or CHO cells. In one embodiment, polypeptides are expressed in E. coli and in baculovirus expression systems. A complete gene can be expressed or, alternatively, fragments of the gene encoding portions of polypeptide can be produced.

In one embodiment, the gene sequence encoding the polypeptide is analyzed to detect putative transmembrane sequences. Such sequences are typically very hydrophobic and are readily detected by the use of standard sequence analysis software, such as MacVector (IBI, New Haven, Conn.). The presence of transmembrane sequences is often deleterious when a recombinant protein is synthesized in many expression systems, especially E. coli, as it leads to the production of insoluble aggregates that are difficult to renature into the native conformation of the protein. Deletion of transmembrane sequences typically does not significantly alter the conformation of the remaining protein structure.

Moreover, transmembrane sequences, being by definition embedded within a membrane, are inaccessible. Therefore, antibodies to these sequences will not prove useful for in vivo or in situ studies. Deletion of transmembrane-encoding sequences from the genes used for expression can be achieved by standard techniques. For example, fortuitously-placed restriction enzyme sites can be used to excise the desired gene fragment, or PCR™-type amplification can be used to amplify only the desired part of the gene. The skilled practitioner will realize that such changes must be designed so as not to change the translational reading frame for downstream portions of the protein-encoding sequence.

In one embodiment, computer sequence analysis is used to determine the location of the predicted major antigenic determinant epitopes of the polypeptide. Software capable of carrying out this analysis is readily available commercially, for example MacVector (IBI, New Haven, Conn.). The software typically uses standard algorithms such as the Kyte/Doolittle or Hopp/Woods methods for locating hydrophilic sequences which are characteristically found on the surface of proteins and are, therefore, likely to act as antigenic determinants.

Once this analysis is made, polypeptides can be prepared that contain at least the essential features of the antigenic determinant and that can be employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants can be constructed and inserted into expression vectors by standard methods, for example, using PCR™ methodology.

The gene or gene fragment encoding a polypeptide can be inserted into an expression vector by standard subcloning techniques. In one embodiment, an E. coli expression vector is used that produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of the protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.).

Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the antigenic ability of the recombinant polypeptide. For example, both the FLAG system and the 6×His system add only short sequences, both of that are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. Other fission systems produce polypeptide where it is desirable to excise the fusion partner from the desired polypeptide. In one embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

Recombinant bacterial cells, for example E. coli, are grown in any of a number of suitable media, for example LB, and the expression of the recombinant polypeptide induced by adding IPTG to the media or switching incubation to a higher temperature. After culturing the bacteria for a further period of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media. The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed.

In another embodiment, the expression system used is one driven by the baculovirus polyhedron promoter. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. One baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant antigen. See Summers et al., A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station.

In designing a gene that encodes a particular polypeptide, the hydropathic index of amino acids may be considered. Table 3 provides a codon table showing the nucleic acids that encode a particular amino acid. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). The following is a brief discussion of the the hydropathic amino acid index for use in the present invention.

TABLE 3 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine −0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

E. Expression of and Delivery of Genes

I. Expression

Once the designer gene, genome or biological system has been made. according the methods described herein, the polynucleotides can be expressed as encoded peptides or proteins of the gene, genome or biological system. The engineering of the polynucleotides for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. Therefore, promoters and other elements specific to a bacterial mammalian or other system may be encluded in the polynucleotide sequence. It is believed that virtually any expression system may be employed in the expression of the claimed nucleic acid sequences.

The artificially generated polynucleotide sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into protein. It is believed that the use of a designer gene version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the designer gene. However, the inventor does not exclude the possibility of employing a genomic version of a particular gene where desired.

As used herein, the terms “engineered” and “recombinant” cells are intended to refer to a cell into which an exogenous polynucleotide described herein has been introduced. Therefore, engineered cells are distinguishable from naturally-occurring cells which do not contain a recombinantly introduced exogenous polynucleotide. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinant cells include those having an introduced polynucleotides, and also include polynucleotides positioned adjacent to a promoter not naturally associated with the particular introduced gene.

To express a recombinant encoded protein or peptide, whether mutant or wild-type, in accordance with the present invention one would prepare an expression vector that comprises one of the claimed isolated nucleic acids under the control of one or more promoters. To bring a coding sequence “under the control of” a promoter, one positions the 5′ end of the translational initiation site of the reading frame generally between about 1 and 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream” promoter stimulates transcription of the inserted DNA and promotes expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in the context used here.

Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli χ 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using pBR322, a plasmid derived from an E. coli species. Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters that can be used by the microbial organism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM™-11 may be utilized in making a recombinant phage vector that can be used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, or the like.

Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.

In a useful insect system, Autograph californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The isolated nucleic acid coding sequences are cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of the coding sequences results in the inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051).

Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cell lines. In addition, a host cell may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the encoded protein.

Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

Specific initiation signals may also be required for efficient translation of the claimed isolated nucleic acid coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this need and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators (Bittner et al., 1987).

In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides “downstream” of the termination site of the protein at a position prior to transcription termination.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding proteins may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines.

It is contemplated that the nucleic acids of the invention may be “overexpressed”, i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in the recombinant host cell. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural human cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.

II. Delivery

In various embodiments of the invention, the expression construct may comprise a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into the host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986) and adeno-associated viruses. Retroviruses also are attractive gene transfer vehicles (Nicolas and Rubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) and adeno-associated virus (Ridgeway, 1988). Such vectors may be used to (i) transform cell lines in vitro for the purpose of expressing proteins of interest or (ii) to transform cells in vitro or in vivo to provide therapeutic polypeptides in a gene therapy scenario. Herpes simplex virus (HSV) is another attractive candidate, especially where neurotropism is desired. HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings.

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al., recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was co-transfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang etal., 1991).

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

In one embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al., (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNA expression construct or DNA segment into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.

In a further embodiment of the invention, the DNA segment or expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of DNA in vitro has been very successful. Wong et al., (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al., (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al., (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.

In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an organism, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an organism. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues. Anderson et al., U.S. Pat. No. 5,399,346, and incorporated herein in its entirety, disclose ex vivo therapeutic methods.

F. Oligonucleotide Synthesis

Oligonucleotide synthesis is well known to those of skill in the art. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.

Phosphoramidite chemistry (Beaucage, and Lyer, 1992) has become by far the most widely used coupling chemistry for the synthesis of oligonucleotides. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product.

Tetrazole is commonly used for the activation of the nucleoside phosphoramidite monomers. Tetrazole has an acidic proton which presumably protonates the basic nitrogen of the diisopropylamino phosphine group, thus making the diisopropylamino group a leaving group. The negatively charged tetrazolium ion then makes an attack on the trivalent phosphorous, forming a transient phosphorous tetrazolide species. The 5′-OH group of the solid support bound nucleoside then attacks the active trivalent phosphorous species, resulting in the formation of the internucleotide linkage. The trivalent phosphorous is finally oxidized to the pentavalent phosphorous. The US patents listed above describe other activators and solid supports for oligonucleotide synthesis.

High throughput oligonucleotide synthesis can be achieved using a synthesizer. The Genome Science and Technology Center, as one aspect of the automation development effort, recently developed a high throughput large scale oligonucleotide synthesizer. This instrument, denoted the MERMADE, is based on a 96-well plate format and uses robotic control to carry out parallel synthesis on 192 samples (2 96-well plates). This device has been variously described in the literature and in presentations, is generally available in the public domain (licensed from the University of Texas and available on contract from Avantec). The device has gone through various generations with differing operating parameters.

The device may be used to synthesize 192 oligonucleotides simultaneously with 99% success. It has virtually 100% success for oligomers less than 60 bp; operates at 20 mM synthesis levels, and gives a product yield of >99% complete synthesis. Using these systems the inventor has synthesized over 10,000 oligomers used for sequencing, PCR™ amplification and recombinant DNA applications. For most uses, including cloning, synthesis success is sufficient such that post synthesis purification is not required.

Once the genome has been synthesized using the methods of the present invention it may be necessary to screen the sequences for analysis of function. Specifically contemplated by the present inventor are chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991).

The use of combinatorial synthesis and high throughput screening assays are well known to those of skill in the art, e.g. U.S. Pat. Nos. 5,807,754; 5,807,683; 5,804,563; 5,789,162; 5,783,384; 5,770,358; 5,759,779; 5,747,334; 5,686,242; 5,198,346; 5,738,996; 5,733,743; 5,714,320; 5,663,046 (each specifically incorporated herein by reference). These patents teach various aspects of the methods and compositions involved in the assembly and activity analyses of high density arrays of different polysubunits (polynucleotides or polypeptides). As such it is contemplated that the methods and compositions described in the patents listed above may be useful in assay the activity profiles of the compositions of the present invention.

The present invention produces a replication competent polynucleotide. Viruses are naturally occurring replication competent pieces of DNA, to the extent that disclosure regarding viruses may be useful in the context of the present invention, the following is a disclosure of viruses. Researchers note that viruses have evolved to be able to deliver their DNA to various host tissues despite the human body's various defensive mechanisms. For this reason, numerous viral vectors have been designed by researchers seeking to create vehicles for therapeutic gene delivery. Some of the types of viruses that have been engineered are listed below.

II. Adenovirus

Adenovirus is a 36 kB, linear, double-strained DNA virus that allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). Adenovirus DNA does not integrate into the host cell chromosomal because adenoviral DNA can replicate in an episomal manner. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. This means that adenovirus can infect non-dividing cells. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans. This group of viruses can be obtained in high titers, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highly infective.

Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5′-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

The E3 region encodes proteins that appears to be necessary for efficient lysis of Ad infected cells as well as preventing TNF-mediated cytolysis and CTL mediated lysis of infected cells. In general, the E4 region encodes is believed to encode seven proteins, some of which activate the E2 promoter. It has been shown to block host mRNA transport and enhance transport of viral RNA to cytoplasm. Further the E4 product is in part responsible for the decrease in early gene expression seen late in infection. E4 also inhibits E1A and E4 (but not E1B) expression during lytic growth. Some E4 proteins are necessary for efficient DNA replication however the mechanism for this involvement is unknown. E4 is also involved in post-transcriptional events in viral late gene expression; ie., alternative splicing of the tripartite leader in lytic growth. Nevertheless, E4 functions are not absolutely required for DNA replication but their lack will delay replication. Other functions include negative regulation of viral DNA synthesis, induction of sub-nuclear reorganization normally seen during adenovirus infection, and other functions that are necessary for viral replication, late viral mRNA accumulation, and host cell transcriptional shut off.

II. Retroviruses

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA to infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed ψ components is constructed (Mann et al., 1983). When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and ψ sequences is introduced into this cell line (by calcium phosphate precipitation for example), the ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration requires the division of host cells (Paskind et al., 1975).

The retrovirus family includes the subfamilies of the oncoviruses, the lentiviruses and the spumaviruses. Two oncoviruses are Moloney murine leukemia virus (MMLV) and feline leukemia virus (FeLV). The lentiviruses include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Among the murine viruses such as MMLV there is a further classification. Murine viruses may be ecotropic, xenotropic, polytropic or amphotropic. Each class of viruses target different cell surface receptors in order to initiate infection.

Further advances in retroviral vector design and concentration methods have allowed production of amphotropic and xenotropic viruses with titers of 10⁸ to 10⁹ cfu/ml (Bowles et al., 1996; Irwin et al, 1994; Jolly, 1994; Kitten et al., 1997).

Replication defective recombinant retroviruses are not acute pathogens in primates (Chowdhury et al., 1991). They have been successfully applied in cell culture systems to transfer the CFTR gene and generate cAMP-activated Cl⁻ secretion in a variety of cell types including human airway epithelia (Drumm et al., 1990, Olsen et al., 1992; Anderson et al., 1991; Olsen et al., 1993). While there is evidence of immune responses to the viral gag and env proteins, this does not prevent successful readministration of vector (McCormack et al., 1997). Further, since recombinant retroviruses have no expressed gene products other than the transgene, the risk of a host inflammatory response due to viral protein expression is limited (McCormack et al., 1997). As for the concern about insertional mutagenesis, to date there are no examples of insertional mutagenesis arising from any human trial with recombinant retroviral vectors.

More recently, hybrid lentivirus vectors have been described combining elements of human immunodeficiency virus (HIV) (Naldini et al., 1996) or feline immunodeficiency virus (FIV) (Poeschla et al., 1998) and MMLV. These vectors transduce nondividing cells in the CNS (Naldini et al., 1996; Blomer et al., 1997), liver (Kafri et al., 1997), muscle (Kafri et al., 1997) and retina (Miyoshi et al., 1997). However, a recent report in xenograft models of human airway epithelia suggests that in well-differentiated epithelia, gene transfer with VSV-G pseudotyped HIV-based lentivirus is inefficient (Goldman et al., 1997).

III. Adeno-Associated Virus

In addition, AAV possesses several unique features that make it more desirable than the other vectors. Unlike retroviruses, AAV can infect non-dividing cells; wild-type AAV has been characterized by integration, in a site-specific manner, into chromosome 19 of human cells (Kotin and Berns, 1989; Kotin et al., 1990; Kotin et al., 1991; Samulski et al., 1991); and AAV also possesses anti-oncogenic properties (Ostrove et al., 1981; Berns and Giraud, 1996). Recombinant AAV genomes are constructed by molecularly cloning DNA sequences of interest between the AAV ITRs, eliminating the entire coding sequences of the wild-type AAV genome. The AAV vectors thus produced lack any of the coding sequences of wild-type AAV, yet retain the property of stable chromosomal integration and expression of the recombinant genes upon transduction both in vitro and in vivo (Berns, 1990; Berns and Bohensky, 1987; Bertran et al., 1996; Kearns et al., 1996; Ponnazhagan et al., 1997a). Until recently, AAV was believed to infect almost all cell types, and even cross species barriers. However, it now has been determined that AAV infection is receptor-mediated (Ponnazhagan et al., 1996; Mizukami et al., 1996).

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription. The sequence of AAV is provided by Srivastava et al. (1983), and in U.S. Pat. No. 5,252,479 (entire text of which is specifically incorporated herein by reference).

The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, p19 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.

AAV is not associated with any pathologic state in humans. Interestingly, for efficient replication, AAV requires “helping” functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many “early” functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.

IV. Vaccinia Virus

Vaccinia viruses are a genus of the poxvirus family. Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA genome of about 186 kB that exhibits a marked “A-T” preference. Inverted terminal repeats of about 10.5 kB flank the genome. The majority of essential genes appear to map within the central region, which is most highly conserved among poxviruses. Estimated open reading frames in vaccinia virus number from 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common. U.S. Pat. No. 5,656,465 (specifically incorporated by reference) describes in vivo gene delivery using pox viruses.

V. Papovavirus

The papovavirus family includes the papillomaviruses and the polyomaviruses. The polyomaviruses include Simian Virus 40 (SV40), polyoma virus and the human polyomaviruses BKV and JCV. Papillomaviruses include the bovine and human papillomaviruses. The genomes of polyomaviruses are circular DNAs of a little more than 5000 bases. The predominant gene products are three virion proteins (VP1-3) and Large T and Small T antigens. Some have an additional structural protein, the agnoprotein, and others have a Middle T antigen. Papillomaviruses are somewhat larger, approaching 8 kB

Little is known about the cellular receptors for polyomaviruses, but polyoma infection can be blocked by treating with sialidase. SV40 will still infect sialidase-treated cells, but JCV cannot hemagglutinate cells treated with sialidase. Because interaction of polyoma VP1 with the cell surface activates c-myc and c-fos, it has been hypothesized that the virus receptor may have some properties of a growth factor receptor. Papillomaviruses are specifically tropic for squamous epithelia, though the specific receptor has not been identified.

VI. Paramyxovirus

The paramyxovirus family is divided into three genera: paramyxovirus, morbillivirus and pneumovirus. The paramyxovirus genus includes the mumps virus and Sendai virus, among others, while the morbilliviruses include the measles virus and the pneumoviruses include respiratory syncytial virus (RSV). Paramyxovirus genomes are RNA based and contain a set of six or more genes, covalently linked in tandem. The genome is something over 15 kB in length. The viral particle is 150-250 nm in diameter, with “fuzzy” projections or spikes protruding therefrom. These are viral glycoproteins that help mediate attachment and entry of the virus into host cells.

A specialized series of proteins are involved in the binding an entry of paramyxoviruses. Attachment in Paramyxoviruses and Morbilliviruses is mediated by glycoproteins that bind to sialic acid-containing receptors. Other proteins anchor the virus by embedding hydrophobic regions in the lipid bilayer of the cell's surface, and exhibit hemagluttinating and neuraminidase activities. In Pnemoviruses, the glycoproptein is heavily glycosylated with O-glycosidic bonds. This molecule lacks the exhibit hemagluttinating and neuraminidase activities of its relatives.

VII. Herpesvirus

Because herpes simplex virus (HSV) is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non-dividing neuronal cells without integrating in to the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency makes HSV an attractive vector. And though much attention has focused on the neurotropic applications of HSV, this vector also can be exploited for other tissues given its wide host range.

Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see Glorioso et al. (1995).

HSV, designated with subtypes 1 and 2, are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. The large, complex, double-stranded DNA genome encodes for dozens of different gene products, some of which derive from spliced transcripts. In addition to virion and envelope structural components, the virus encodes numerous other proteins including a protease, a ribonucleotides reductase, a DNA polymerase, a ssDNA binding protein, a helicase/primase, a DNA dependent ATPase, a dUTPase and others.

HSV genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion (Honess and Roizrnan, 1974; Honess and Roizman 1975; Roizman and Sears, 1995). The expression of a genes, the first set of genes to be expressed after infection, is enhanced by the virion protein number 16, or α-transducing factor (Post et al., 1981; Batterson and Roizman, 1983; Campbell et al., 1983). The expression of β genes requires functional α gene products, most notably ICP4, which is encoded by the α4 gene (DeLuca et al., 1985). γ genes, a heterogeneous group of genes encoding largely virion structural proteins, require the onset of viral DNA synthesis for optimal expression (Holland et al., 1980).

In line with the complexity of the genome, the life cycle of HSV is quite involved. In addition to the lytic cycle, which results in synthesis of virus particles and, eventually, cell death, the virus has the capability to enter a latent state in which the genome is maintained in neural ganglia until some as of yet undefined signal triggers a recurrence of the lytic cycle. Avirulent variants of HSV have been developed and are readily available for use in gene therapy contexts (U.S. Pat. No. 5,672,344).

G. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Combinatoric Gene Assembly

The inventor has developed a strategy of oligomer assembly into larger DNA molecules denoted combinatoric assembly. The procedure is carried out as follows: one may design a plasmid using one of a number of commercial or public domain computer programs to contain the genes, promoters, drug selection, origin of replication, etc. required. SynGene v.2.0 is a program that generates a list of overlapping oligonucleotides sufficient to reassemble the gene or plasmid (see FIG. 7). For instance, for a 5000 bp gene, SynGene 2.0 can generate two lists of 100 component 50 mers from one strand and 100 component 50 mers from the complementary strand such that each pair of oligomers will overlap by 25 base pairs. The program checks the sequence for repeats and produces a MERMADE input file which directly programs the oligonucleotide synthesizer. The synthesizer produces two sets of 96-well plates containing the complementary oligonucleotides. A SynGene program is depicted in FIG. 7. This program is designed to break down a designer gene or genome into oligonucleotides fore synthesis. The program is for the complete synthetic designer gene and is based upon an original program for formatting DNA sequences written by Dr. Glen Evans.

Combinatoric assembly is best carried out using a programmable robotic workstation such as a Beckman Biomek 2000. In short, pairs of oligomers which overlap are mixed and annealed. Following annealing, a smaller set of duplex oligomers is generated. These are again paired and annealed, forming a smaller set of larger oligomers. Sequentially, overlapping oligomers are allowed to anneal until the entire reassembly is completed. Annealing may be carried out in the absence of ligase, or each step may be followed by ligation. In one configuration, oligomers are annealed in the presence of topoisomerase 2, which does not require 5′ phosphorylation of the oligomer, occurs at room temperature, and is a rapid (5 minute) reaction as opposed to 12 h ligation at 12°. Following the complete assembly, the resulting DNA molecule can be used for its designed purpose, usually transformation into a bacterial host for replication. The steps in this cycle are outlined in FIG. 3.

This approach has a major advantage over traditional recombinant DNA based cloning. While it is technically feasible to make virtually any modification or mutation in existing DNA molecules, the effort required, as will as the high technical skill, make some constructions difficult or tedious. This method, while having been used for many years, is not applicable to automated gene cloning or large scale creation or entirely novel DNA sequences.

Example 2 Production of Artificial Genes

In one example, the present invention will produce a known gene of about 1000 base pairs in length by the following method. A set of oligonucleotides, each of 50 bases, is generated such that the entire plus strand of the gene is represented. A second set of oligonucleotides, also comprised of 50-mers, is generated for the minus strand. This set is designed, however, such that complementary pairing with the first and second sets results in overlap of “paired” sequences, i.e., each oligonucleotide of the first set is complementary with regions from two oligonucleotides of the second set (with the possible exception of the terminal oligonucleotides). The region of overlap is set at 30 bases, leaving a 20 base pair overhang for each pair. The first and said second set of oligonucleotides is annealed in a single mixture and treated with a ligating enzyme.

In another example, the gene to be synthesized is about 5000 base pairs. Each set of oligonucleotides is made up of fifty 100-mers with overlapping regions, of complementary oligonucleotides, of 75 bases, leaving 25 base “sticky ends.” In this embodiment, the 5′ terminal oligonucleotide of the first oligonucleotide set is annealed with the 3′ terminal oligonucleotide of the second set to form a first annealed product, then the next most 5′ terminal oligonucleotide of the first set is annealed with the first annealed product to form a second annealed product, and the process is repeated until all oligonucleotides of said first and said second sets have been annealed. Ligation of the products may occur between steps or at the conclusion of all hybridizations.

In a third example, a gene of 100,000 bp is synthesize from one thousand 100-mers. Again, the overlap between “pairs” of plus and minus oligonucleotides is 75 bases, leaving a 25 base pair overhang. In this method, a combinatorial approach is used where corresponding pairs of partially complementary oligonucleotides are hybridized in first step. A second round of hybridization then is undertaken with appropriately complementary pairs of products from the first round. This process is repeated a total of 10 times, each round of hybridization reducing the number of products by half. Ligation of the products then is performed.

Example 3 Large Scale Expression of Human Gene Products

Once the human genome has been characterized, functional analysis of the human genome, based upon the complete sequence, will require a variety of approaches to structural, functional and network biology. The approach proposed herein for producing a series of expression constructs representing all potential human gene products and the assembly of sets of bacterial and/or yeast expressing these products will provide an important avenue into the beginnings of functional analysis.

Secondly, the approach described here, when developed to its theoretical optima, will allow the large scale transfer of genes to cell lines or organisms for functional analysis. The long term goal of this concept is the creation of living organisms entirely based on bioinformatics and information processing. Obviously, the knowledge of the complete sequence is not sufficient to appreciate the myriad of biological concepts inherent in life.

Example 4 Construction of a Synthetic Plasmid

A DNA molecule was designed using synthetic parts of previously known plasmids. As a demonstration of this technique, plasmid synlux4 was designed. Synlux4 consists of 4800 base pairs of DNA. Within this sequence are included the sequence of lux A and lx B, the A and B components of the luciferase protein from Vibrio Fisheri, potions of plasmid pUC19 including the origin of replication and replication stability sequences, the promoter and coding sequence for tn9 kanamnycin/neomycin phosphotransferase. The sequence was designed on a computer using Microsoft Word and Vector NTI (InforMax, Inc.). The sequence is listed in FIG. 4.

Following design, a computer program SynGene 2.0 was used to break the sequence down into components consisting of overlapping 50-mer oligonucleotides. From the 4800 base pair sequence, 192 50-mers were designed. The component oligonucleotides are listed in FIG. 5. These component oligonucleotides were synthesized using a custom 96-well oligonucleotide synthesizer (Rayner, et al.) Genome Research, 8, 741-747 (1998). The component oligonucleotides were produced in two 96-well microtitre plates, each plate holding one set of component oligonucleotides. Thus, plate one held the forward strand oligos and plate 2 held the reverse strand oligos.

The oligonucleotides were assembled and ligations carried out using a Biomek 1000 robotic workstation (Beckman). Sequential transfers of oligonucleotides were done by pipetting from one well to a second well of the plate and a ligation reaction carried out using T4 ligase. The pattern of assembly is delineated in FIG. 6.

Following assembly, the resulting ligation mix was used to transform competent E. coli strain DH5a. The transformation mix was plated on LB plates containing 25 μg/ml kanamycin sulfate. and recombinant colonies obtained. The resulting recombinant clones were isolated, cloned, and DNA prepared. The DNA was analyzed on 1% agarose gels in order detect recombinant molecules. Clones were shown to contain the expected 4800 base pair plasmid containing lux A and B genes.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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193 1 4800 DNA Artificial Sequence Synthetic plasmid 1 aagcttacct cgatttgagg acgttacaag tattactgtt aaggagcgta gattaaaaaa 60 tgaaattgaa aatgaattat tagaattggc ttaaataaac agaatcacca aaaaggaata 120 gagtatgaag tttggaaata tttgtttttc gtatcaacca ccaggtgaaa ctcataagct 180 aagtaatgga tcgctttgtt cggcttggta tcgcctcaga agagtagggt ttgatacata 240 ttggacctta gaacatcatt ttacagagtt tggtcttacg ggaaatttat ttgttgctgc 300 ggctaacctg ttaggaagaa ctaaaacatt aaatgttggc actatggggg ttgttattcc 360 gacagcacac ccagttcgac agttagaaga cgttttatta ttagatcaaa tgtcgaaagg 420 tcgttttaat tttggaaccg ttcgagggct ataccataaa gattttcgag tatttggtgt 480 tgatatggaa gagtctcgag caattactca aaatttctac cagatgataa tggaaagctt 540 acagacagga accattagct ctgatagtga ttacattcaa tttcctaagg ttgatgtata 600 tcccaaagtg tactcaaaaa atgtaccaac ctgtatgact gctgagtccg caagtacgac 660 agaatggcta gcaatacaag ggctaccaat ggttcttagt tggattattg gtactaatga 720 aaaaaaagca cagatggaac tctataatga aattgcgaca gaatatggtc atgatatatc 780 taaaatagat cattgtatga cttatatttg ttctgttgat gatgatgcac aaaaggcgca 840 agatgtttgt cgggagtttc tgaaaaattg gtatgactca tatgtaaatg cgaccaatat 900 ctttaatgat agcaatcaaa ctcgtggtta tgattatcat aaaggtcaat ggcgtgattt 960 tgttttacaa ggacatacaa acaccaatcg acgtgttgat tatagcaatg gtattaaccc 1020 tgtaggcact cctgagcagt gtattgaaat cattcaacgt gatattgatg caacgggtat 1080 tacaaacatt acatgcggat ttgaagctaa tggaactgaa gatgaaataa ttgcttccat 1140 gcgacgcttt atgacacaag tcgctccttt cttaaaagaa cctaaataaa ttacttattt 1200 gatactagag ataataagga acaagttatg aaatttggat tattttttct aaactttcag 1260 aaagatggaa taacatctga agaaacgttg gataatatgg taaagactgt cacgttaatt 1320 gattcaacta aatatcattt taatactgcc tttgttaatg aacatcactt ttcaaaaaat 1380 ggtattgttg gagcacctat taccgcagct ggttttttat tagggttaac aaataaatta 1440 catattggtt cattaaatca agtaattacc acccatcacc ctgtacgtgt agcagaagaa 1500 gccagtttat tagatcaaat gtcagaggga cgcttcattc ttggttttag tgactgcgaa 1560 agtgatttcg aaatggaatt ttttagacgt catatctcat caaggcaaca acaatttgaa 1620 gcatgctatg aaataattaa tgacgcatta actacaggtt attgtcatcc ccaaaacgac 1680 ttttatgatt ttccaaaggt ttcaattaat ccacactgtt acagtgagaa tggacctaag 1740 caatatgtat ccgctacatc aaaagaagtc gtcatgtggg cagcgaaaaa ggcactgcct 1800 ttaacattta agtgggagga taatttagaa accaaagaac gctatgcaat tctatataat 1860 aaaacagcac aacaatatgg tattgatatt tcggatgttg atcatcaatt aactgtaatt 1920 gcgaacttaa atgctgatag aagtacggct caagaagaag tgagagaata cttaaaagac 1980 tatatcactg aaacttaccc tcaaatggac agagatgaaa aaattaactg cattattgaa 2040 gagaatgcag ttgggtctca tgatgactat tatgaatcga caaaattagc agtggaaaaa 2100 acagggtcta aaaatatttt attatccttt gaatcaatgt ccgatattaa agatgtaaaa 2160 gatattattg atatgttgaa ccaaaaaatc gaaatgaatt taccataata aaattaaagg 2220 caatttctat attagattgc ctttttgggg atcctctaga aatattttat ctgattaata 2280 agatgagaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 2340 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 2400 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 2460 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 2520 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 2580 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2640 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 2700 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 2760 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaaaagcttc 2820 acgctgccgc aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc 2880 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag 2940 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct 3000 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg 3060 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg 3120 gcgcagggga tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca 3180 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 3240 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 3300 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc 3360 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 3420 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 3480 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 3540 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 3600 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 3660 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct 3720 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 3780 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 3840 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 3900 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 3960 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 4020 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 4080 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccggg 4140 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 4200 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 4260 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 4320 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 4380 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 4440 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 4500 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 4560 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 4620 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 4680 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 4740 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 4800 2 50 DNA Artificial Sequence Synthetic Oligonucleotide 2 aagcttacct cgatttgagg acgttacaag tattactgtt aaggagcgta 50 3 50 DNA Artificial Sequence Synthetic Oligonucleotide 3 gattaaaaaa tgaaattgaa aatgaattat tagaattggc ttaaataaac 50 4 50 DNA Artificial Sequence Synthetic Oligonucleotide 4 agaatcacca aaaaggaata gagtatgaag tttggaaata tttgtttttc 50 5 50 DNA Artificial Sequence Synthetic Oligonucleotide 5 gtatcaacca ccaggtgaaa ctcataagct aagtaatgga tcgctttgtt 50 6 50 DNA Artificial Sequence Synthetic Oligonucleotide 6 cggcttggta tcgcctcaga agagtagggt ttgatacata ttggacctta 50 7 50 DNA Artificial Sequence Synthetic Oligonucleotide 7 gaacatcatt ttacagagtt tggtcttacg ggaaatttat ttgttgctgc 50 8 50 DNA Artificial Sequence Synthetic Oligonucleotide 8 ggctaacctg ttaggaagaa ctaaaacatt aaatgttggc actatggggg 50 9 50 DNA Artificial Sequence Synthetic Oligonucleotide 9 ttgttattcc gacagcacac ccagttcgac agttagaaga cgttttatta 50 10 50 DNA Artificial Sequence Synthetic Oligonucleotide 10 ttagatcaaa tgtcgaaagg tcgttttaat tttggaaccg ttcgagggct 50 11 50 DNA Artificial Sequence Synthetic Oligonucleotide 11 ataccataaa gattttcgag tatttggtgt tgatatggaa gagtctcgag 50 12 50 DNA Artificial Sequence Synthetic Oligonucleotide 12 caattactca aaatttctac cagatgataa tggaaagctt acagacagga 50 13 50 DNA Artificial Sequence Synthetic Oligonucleotide 13 accattagct ctgatagtga ttacattcaa tttcctaagg ttgatgtata 50 14 50 DNA Artificial Sequence Synthetic Oligonucleotide 14 tcccaaagtg tactcaaaaa atgtaccaac ctgtatgact gctgagtccg 50 15 50 DNA Artificial Sequence Synthetic Oligonucleotide 15 caagtacgac agaatggcta gcaatacaag ggctaccaat ggttcttagt 50 16 50 DNA Artificial Sequence Synthetic Oligonucleotide 16 tggattattg gtactaatga aaaaaaagca cagatggaac tctataatga 50 17 50 DNA Artificial Sequence Synthetic Oligonucleotide 17 aattgcgaca gaatatggtc atgatatatc taaaatagat cattgtatga 50 18 50 DNA Artificial Sequence Synthetic Oligonucleotide 18 cttatatttg ttctgttgat gatgatgcac aaaaggcgca agatgtttgt 50 19 50 DNA Artificial Sequence Synthetic Oligonucleotide 19 cgggagtttc tgaaaaattg gtatgactca tatgtaaatg cgaccaatat 50 20 50 DNA Artificial Sequence Synthetic Oligonucleotide 20 ctttaatgat agcaatcaaa ctcgtggtta tgattatcat aaaggtcaat 50 21 50 DNA Artificial Sequence Synthetic Oligonucleotide 21 ggcgtgattt tgttttacaa ggacatacaa acaccaatcg acgtgttgat 50 22 50 DNA Artificial Sequence Synthetic Oligonucleotide 22 tatagcaatg gtattaaccc tgtaggcact cctgagcagt gtattgaaat 50 23 50 DNA Artificial Sequence Synthetic Oligonucleotide 23 cattcaacgt gatattgatg caacgggtat tacaaacatt acatgcggat 50 24 50 DNA Artificial Sequence Synthetic Oligonucleotide 24 ttgaagctaa tggaactgaa gatgaaataa ttgcttccat gcgacgcttt 50 25 50 DNA Artificial Sequence Synthetic Oligonucleotide 25 atgacacaag tcgctccttt cttaaaagaa cctaaataaa ttacttattt 50 26 50 DNA Artificial Sequence Synthetic Oligonucleotide 26 gatactagag ataataagga acaagttatg aaatttggat tattttttct 50 27 50 DNA Artificial Sequence Synthetic Oligonucleotide 27 aaactttcag aaagatggaa taacatctga agaaacgttg gataatatgg 50 28 50 DNA Artificial Sequence Synthetic Oligonucleotide 28 taaagactgt cacgttaatt gattcaacta aatatcattt taatactgcc 50 29 50 DNA Artificial Sequence Synthetic Oligonucleotide 29 tttgttaatg aacatcactt ttcaaaaaat ggtattgttg gagcacctat 50 30 50 DNA Artificial Sequence Synthetic Oligonucleotide 30 taccgcagct ggttttttat tagggttaac aaataaatta catattggtt 50 31 50 DNA Artificial Sequence Synthetic Oligonucleotide 31 cattaaatca agtaattacc acccatcacc ctgtacgtgt agcagaagaa 50 32 50 DNA Artificial Sequence Synthetic Oligonucleotide 32 gccagtttat tagatcaaat gtcagaggga cgcttcattc ttggttttag 50 33 50 DNA Artificial Sequence Synthetic Oligonucleotide 33 tgactgcgaa agtgatttcg aaatggaatt ttttagacgt catatctcat 50 34 50 DNA Artificial Sequence Synthetic Oligonucleotide 34 caaggcaaca acaatttgaa gcatgctatg aaataattaa tgacgcatta 50 35 50 DNA Artificial Sequence Synthetic Oligonucleotide 35 actacaggtt attgtcatcc ccaaaacgac ttttatgatt ttccaaaggt 50 36 50 DNA Artificial Sequence Synthetic Oligonucleotide 36 ttcaattaat ccacactgtt acagtgagaa tggacctaag caatatgtat 50 37 50 DNA Artificial Sequence Synthetic Oligonucleotide 37 ccgctacatc aaaagaagtc gtcatgtggg cagcgaaaaa ggcactgcct 50 38 50 DNA Artificial Sequence Synthetic Oligonucleotide 38 ttaacattta agtgggagga taatttagaa accaaagaac gctatgcaat 50 39 50 DNA Artificial Sequence Synthetic Oligonucleotide 39 tctatataat aaaacagcac aacaatatgg tattgatatt tcggatgttg 50 40 50 DNA Artificial Sequence Synthetic Oligonucleotide 40 atcatcaatt aactgtaatt gcgaacttaa atgctgatag aagtacggct 50 41 50 DNA Artificial Sequence Synthetic Oligonucleotide 41 caagaagaag tgagagaata cttaaaagac tatatcactg aaacttaccc 50 42 50 DNA Artificial Sequence Synthetic Oligonucleotide 42 tcaaatggac agagatgaaa aaattaactg cattattgaa gagaatgcag 50 43 50 DNA Artificial Sequence Synthetic Oligonucleotide 43 ttgggtctca tgatgactat tatgaatcga caaaattagc agtggaaaaa 50 44 50 DNA Artificial Sequence Synthetic Oligonucleotide 44 acagggtcta aaaatatttt attatccttt gaatcaatgt ccgatattaa 50 45 50 DNA Artificial Sequence Synthetic Oligonucleotide 45 agatgtaaaa gatattattg atatgttgaa ccaaaaaatc gaaatgaatt 50 46 50 DNA Artificial Sequence Synthetic Oligonucleotide 46 taccataata aaattaaagg caatttctat attagattgc ctttttgggg 50 47 50 DNA Artificial Sequence Synthetic Oligonucleotide 47 atcctctaga aatattttat ctgattaata agatgagaat tcactggccg 50 48 50 DNA Artificial Sequence Synthetic Oligonucleotide 48 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 50 49 50 DNA Artificial Sequence Synthetic Oligonucleotide 49 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 50 50 50 DNA Artificial Sequence Synthetic Oligonucleotide 50 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 50 51 50 DNA Artificial Sequence Synthetic Oligonucleotide 51 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 50 52 50 DNA Artificial Sequence Synthetic Oligonucleotide 52 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 50 53 50 DNA Artificial Sequence Synthetic Oligonucleotide 53 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 50 54 50 DNA Artificial Sequence Synthetic Oligonucleotide 54 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 50 55 50 DNA Artificial Sequence Synthetic Oligonucleotide 55 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 50 56 50 DNA Artificial Sequence Synthetic Oligonucleotide 56 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 50 57 50 DNA Artificial Sequence Synthetic Oligonucleotide 57 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 50 58 50 DNA Artificial Sequence Synthetic Oligonucleotide 58 ttatttttct aaaaagcttc acgctgccgc aagcactcag ggcgcaaggg 50 59 50 DNA Artificial Sequence Synthetic Oligonucleotide 59 ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct 50 60 50 DNA Artificial Sequence Synthetic Oligonucleotide 60 gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 50 61 50 DNA Artificial Sequence Synthetic Oligonucleotide 61 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct 50 62 50 DNA Artificial Sequence Synthetic Oligonucleotide 62 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg 50 63 50 DNA Artificial Sequence Synthetic Oligonucleotide 63 cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc 50 64 50 DNA Artificial Sequence Synthetic Oligonucleotide 64 ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac 50 65 50 DNA Artificial Sequence Synthetic Oligonucleotide 65 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 50 66 50 DNA Artificial Sequence Synthetic Oligonucleotide 66 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 50 67 50 DNA Artificial Sequence Synthetic Oligonucleotide 67 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 50 68 50 DNA Artificial Sequence Synthetic Oligonucleotide 68 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc 50 69 50 DNA Artificial Sequence Synthetic Oligonucleotide 69 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 50 70 50 DNA Artificial Sequence Synthetic Oligonucleotide 70 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 50 71 50 DNA Artificial Sequence Synthetic Oligonucleotide 71 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 50 72 50 DNA Artificial Sequence Synthetic Oligonucleotide 72 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 50 73 50 DNA Artificial Sequence Synthetic Oligonucleotide 73 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 50 74 50 DNA Artificial Sequence Synthetic Oligonucleotide 74 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag 50 75 50 DNA Artificial Sequence Synthetic Oligonucleotide 75 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 50 76 50 DNA Artificial Sequence Synthetic Oligonucleotide 76 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 50 77 50 DNA Artificial Sequence Synthetic Oligonucleotide 77 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 50 78 50 DNA Artificial Sequence Synthetic Oligonucleotide 78 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 50 79 50 DNA Artificial Sequence Synthetic Oligonucleotide 79 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 50 80 50 DNA Artificial Sequence Synthetic Oligonucleotide 80 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg 50 81 50 DNA Artificial Sequence Synthetic Oligonucleotide 81 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga 50 82 50 DNA Artificial Sequence Synthetic Oligonucleotide 82 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 50 83 50 DNA Artificial Sequence Synthetic Oligonucleotide 83 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 50 84 50 DNA Artificial Sequence Synthetic Oligonucleotide 84 gcgcggggat ctcatgctgg agttcttcgc ccaccccggg catgaccaaa 50 85 50 DNA Artificial Sequence Synthetic Oligonucleotide 85 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 50 86 50 DNA Artificial Sequence Synthetic Oligonucleotide 86 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 50 87 50 DNA Artificial Sequence Synthetic Oligonucleotide 87 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 50 88 50 DNA Artificial Sequence Synthetic Oligonucleotide 88 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 50 89 51 DNA Artificial Sequence Synthetic Oligonucleotide 89 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaatg a 51 90 50 DNA Artificial Sequence Synthetic Oligonucleotide 90 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 50 91 50 DNA Artificial Sequence Synthetic Oligonucleotide 91 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 50 92 50 DNA Artificial Sequence Synthetic Oligonucleotide 92 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 50 93 50 DNA Artificial Sequence Synthetic Oligonucleotide 93 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 50 94 50 DNA Artificial Sequence Synthetic Oligonucleotide 94 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 50 95 50 DNA Artificial Sequence Synthetic Oligonucleotide 95 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 50 96 50 DNA Artificial Sequence Synthetic Oligonucleotide 96 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 50 97 50 DNA Artificial Sequence Synthetic Oligonucleotide 97 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 50 98 50 DNA Artificial Sequence Synthetic Oligonucleotide 98 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 50 99 50 DNA Artificial Sequence Synthetic Oligonucleotide 99 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 50 100 50 DNA Artificial Sequence Synthetic Oligonucleotide 100 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 50 101 50 DNA Artificial Sequence Synthetic Oligonucleotide 101 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 50 102 50 DNA Artificial Sequence Synthetic Oligonucleotide 102 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 50 103 50 DNA Artificial Sequence Synthetic Oligonucleotide 103 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 50 104 50 DNA Artificial Sequence Synthetic Oligonucleotide 104 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 50 105 50 DNA Artificial Sequence Synthetic Oligonucleotide 105 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 50 106 50 DNA Artificial Sequence Synthetic Oligonucleotide 106 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 50 107 50 DNA Artificial Sequence Synthetic Oligonucleotide 107 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 50 108 50 DNA Artificial Sequence Synthetic Oligonucleotide 108 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 50 109 50 DNA Artificial Sequence Synthetic Oligonucleotide 109 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 50 110 50 DNA Artificial Sequence Synthetic Oligonucleotide 110 tggaacgaaa actcacgtta agggattttg gtcatgcccg gggtgggcga 50 111 50 DNA Artificial Sequence Synthetic Oligonucleotide 111 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc 50 112 50 DNA Artificial Sequence Synthetic Oligonucleotide 112 cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc 50 113 50 DNA Artificial Sequence Synthetic Oligonucleotide 113 gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc 50 114 50 DNA Artificial Sequence Synthetic Oligonucleotide 114 ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc 50 115 50 DNA Artificial Sequence Synthetic Oligonucleotide 115 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc 50 116 50 DNA Artificial Sequence Synthetic Oligonucleotide 116 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc 50 117 50 DNA Artificial Sequence Synthetic Oligonucleotide 117 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 50 118 50 DNA Artificial Sequence Synthetic Oligonucleotide 118 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc 50 119 50 DNA Artificial Sequence Synthetic Oligonucleotide 119 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag 50 120 50 DNA Artificial Sequence Synthetic Oligonucleotide 120 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga 50 121 50 DNA Artificial Sequence Synthetic Oligonucleotide 121 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct 50 122 50 DNA Artificial Sequence Synthetic Oligonucleotide 122 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat 50 123 50 DNA Artificial Sequence Synthetic Oligonucleotide 123 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 50 124 50 DNA Artificial Sequence Synthetic Oligonucleotide 124 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct 50 125 50 DNA Artificial Sequence Synthetic Oligonucleotide 125 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag 50 126 50 DNA Artificial Sequence Synthetic Oligonucleotide 126 ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca 50 127 50 DNA Artificial Sequence Synthetic Oligonucleotide 127 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac 50 128 50 DNA Artificial Sequence Synthetic Oligonucleotide 128 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa 50 129 50 DNA Artificial Sequence Synthetic Oligonucleotide 129 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 50 130 50 DNA Artificial Sequence Synthetic Oligonucleotide 130 caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc 50 131 50 DNA Artificial Sequence Synthetic Oligonucleotide 131 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca 50 132 50 DNA Artificial Sequence Synthetic Oligonucleotide 132 gggcttccca accttaccag agggcgcccc agctggcaat tccggttcgc 50 133 50 DNA Artificial Sequence Synthetic Oligonucleotide 133 ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg 50 134 50 DNA Artificial Sequence Synthetic Oligonucleotide 134 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc 50 135 50 DNA Artificial Sequence Synthetic Oligonucleotide 135 ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt 50 136 50 DNA Artificial Sequence Synthetic Oligonucleotide 136 tctacgtgtt ccgcttcctt tagcagccct tgcgccctga gtgcttgcgg 50 137 50 DNA Artificial Sequence Synthetic Oligonucleotide 137 cagcgtgaag ctttttagaa aaataaacaa ataggggttc cgcgcacatt 50 138 50 DNA Artificial Sequence Synthetic Oligonucleotide 138 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 50 139 50 DNA Artificial Sequence Synthetic Oligonucleotide 139 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc 50 140 50 DNA Artificial Sequence Synthetic Oligonucleotide 140 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 50 141 50 DNA Artificial Sequence Synthetic Oligonucleotide 141 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 50 142 50 DNA Artificial Sequence Synthetic Oligonucleotide 142 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 50 143 50 DNA Artificial Sequence Synthetic Oligonucleotide 143 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg 50 144 50 DNA Artificial Sequence Synthetic Oligonucleotide 144 cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca 50 145 50 DNA Artificial Sequence Synthetic Oligonucleotide 145 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 50 146 50 DNA Artificial Sequence Synthetic Oligonucleotide 146 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 50 147 50 DNA Artificial Sequence Synthetic Oligonucleotide 147 ttcccagtca cgacgttgta aaacgacggc cagtgaattc tcatcttatt 50 148 50 DNA Artificial Sequence Synthetic Oligonucleotide 148 aatcagataa aatatttcta gaggatcccc aaaaaggcaa tctaatatag 50 149 50 DNA Artificial Sequence Synthetic Oligonucleotide 149 aaattgcctt taattttatt atggtaaatt catttcgatt ttttggttca 50 150 50 DNA Artificial Sequence Synthetic Oligonucleotide 150 acatatcaat aatatctttt acatctttaa tatcggacat tgattcaaag 50 151 50 DNA Artificial Sequence Synthetic Oligonucleotide 151 gataataaaa tatttttaga ccctgttttt tccactgcta attttgtcga 50 152 50 DNA Artificial Sequence Synthetic Oligonucleotide 152 ttcataatag tcatcatgag acccaactgc attctcttca ataatgcagt 50 153 50 DNA Artificial Sequence Synthetic Oligonucleotide 153 taattttttc atctctgtcc atttgagggt aagtttcagt gatatagtct 50 154 50 DNA Artificial Sequence Synthetic Oligonucleotide 154 tttaagtatt ctctcacttc ttcttgagcc gtacttctat cagcatttaa 50 155 50 DNA Artificial Sequence Synthetic Oligonucleotide 155 gttcgcaatt acagttaatt gatgatcaac atccgaaata tcaataccat 50 156 50 DNA Artificial Sequence Synthetic Oligonucleotide 156 attgttgtgc tgttttatta tatagaattg catagcgttc tttggtttct 50 157 50 DNA Artificial Sequence Synthetic Oligonucleotide 157 aaattatcct cccacttaaa tgttaaaggc agtgcctttt tcgctgccca 50 158 50 DNA Artificial Sequence Synthetic Oligonucleotide 158 catgacgact tcttttgatg tagcggatac atattgctta ggtccattct 50 159 50 DNA Artificial Sequence Synthetic Oligonucleotide 159 cactgtaaca gtgtggatta attgaaacct ttggaaaatc ataaaagtcg 50 160 50 DNA Artificial Sequence Synthetic Oligonucleotide 160 ttttggggat gacaataacc tgtagttaat gcgtcattaa ttatttcata 50 161 50 DNA Artificial Sequence Synthetic Oligonucleotide 161 gcatgcttca aattgttgtt gccttgatga gatatgacgt ctaaaaaatt 50 162 50 DNA Artificial Sequence Synthetic Oligonucleotide 162 ccatttcgaa atcactttcg cagtcactaa aaccaagaat gaagcgtccc 50 163 50 DNA Artificial Sequence Synthetic Oligonucleotide 163 tctgacattt gatctaataa actggcttct tctgctacac gtacagggtg 50 164 50 DNA Artificial Sequence Synthetic Oligonucleotide 164 atgggtggta attacttgat ttaatgaacc aatatgtaat ttatttgtta 50 165 50 DNA Artificial Sequence Synthetic Oligonucleotide 165 accctaataa aaaaccagct gcggtaatag gtgctccaac aataccattt 50 166 50 DNA Artificial Sequence Synthetic Oligonucleotide 166 tttgaaaagt gatgttcatt aacaaaggca gtattaaaat gatatttagt 50 167 50 DNA Artificial Sequence Synthetic Oligonucleotide 167 tgaatcaatt aacgtgacag tctttaccat attatccaac gtttcttcag 50 168 50 DNA Artificial Sequence Synthetic Oligonucleotide 168 atgttattcc atctttctga aagtttagaa aaaataatcc aaatttcata 50 169 50 DNA Artificial Sequence Synthetic Oligonucleotide 169 acttgttcct tattatctct agtatcaaat aagtaattta tttaggttct 50 170 50 DNA Artificial Sequence Synthetic Oligonucleotide 170 tttaagaaag gagcgacttg tgtcataaag cgtcgcatgg aagcaattat 50 171 50 DNA Artificial Sequence Synthetic Oligonucleotide 171 ttcatcttca gttccattag cttcaaatcc gcatgtaatg tttgtaatac 50 172 50 DNA Artificial Sequence Synthetic Oligonucleotide 172 ccgttgcatc aatatcacgt tgaatgattt caatacactg ctcaggagtg 50 173 50 DNA Artificial Sequence Synthetic Oligonucleotide 173 cctacagggt taataccatt gctataatca acacgtcgat tggtgtttgt 50 174 50 DNA Artificial Sequence Synthetic Oligonucleotide 174 atgtccttgt aaaacaaaat cacgccattg acctttatga taatcataac 50 175 50 DNA Artificial Sequence Synthetic Oligonucleotide 175 cacgagtttg attgctatca ttaaagatat tggtcgcatt tacatatgag 50 176 50 DNA Artificial Sequence Synthetic Oligonucleotide 176 tcataccaat ttttcagaaa ctcccgacaa acatcttgcg ccttttgtgc 50 177 50 DNA Artificial Sequence Synthetic Oligonucleotide 177 atcatcatca acagaacaaa tataagtcat acaatgatct attttagata 50 178 50 DNA Artificial Sequence Synthetic Oligonucleotide 178 tatcatgacc atattctgtc gcaatttcat tatagagttc catctgtgct 50 179 50 DNA Artificial Sequence Synthetic Oligonucleotide 179 tttttttcat tagtaccaat aatccaacta agaaccattg gtagcccttg 50 180 50 DNA Artificial Sequence Synthetic Oligonucleotide 180 tattgctagc cattctgtcg tacttgcgga ctcagcagtc atacaggttg 50 181 50 DNA Artificial Sequence Synthetic Oligonucleotide 181 gtacattttt tgagtacact ttgggatata catcaacctt aggaaattga 50 182 50 DNA Artificial Sequence Synthetic Oligonucleotide 182 atgtaatcac tatcagagct aatggttcct gtctgtaagc tttccattat 50 183 50 DNA Artificial Sequence Synthetic Oligonucleotide 183 catctggtag aaattttgag taattgctcg agactcttcc atatcaacac 50 184 50 DNA Artificial Sequence Synthetic Oligonucleotide 184 caaatactcg aaaatcttta tggtatagcc ctcgaacggt tccaaaatta 50 185 50 DNA Artificial Sequence Synthetic Oligonucleotide 185 aaacgacctt tcgacatttg atctaataat aaaacgtctt ctaactgtcg 50 186 50 DNA Artificial Sequence Synthetic Oligonucleotide 186 aactgggtgt gctgtcggaa taacaacccc catagtgcca acatttaatg 50 187 50 DNA Artificial Sequence Synthetic Oligonucleotide 187 ttttagttct tcctaacagg ttagccgcag caacaaataa atttcccgta 50 188 50 DNA Artificial Sequence Synthetic Oligonucleotide 188 agaccaaact ctgtaaaatg atgttctaag gtccaatatg tatcaaaccc 50 189 50 DNA Artificial Sequence Synthetic Oligonucleotide 189 tactcttctg aggcgatacc aagccgaaca aagcgatcca ttacttagct 50 190 50 DNA Artificial Sequence Synthetic Oligonucleotide 190 tatgagtttc acctggtggt tgatacgaaa aacaaatatt tccaaacttc 50 191 50 DNA Artificial Sequence Synthetic Oligonucleotide 191 atactctatt cctttttggt gattctgttt atttaagcca attctaataa 50 192 50 DNA Artificial Sequence Synthetic Oligonucleotide 192 ttcattttca atttcatttt ttaatctacg ctccttaaca gtaatacttg 50 193 50 DNA Artificial Sequence Synthetic Oligonucleotide 193 taacgtcctc aaatcgaggt aagcttcata ggctccgccc ccctgacgag 50 

What is claimed is:
 1. A method of synthesizing a double-stranded polynucleotide, comprising: (a) annealing a 5′ terminal oligonucleotide with a 3′ terminal oligonucleotide of a double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal oligonucleotide of said double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, said next most terminal oligonucleotide having a length of at least about 25 bases, and (b) repeating step (a) one or more times to sequentially produce a double-stranded polynucleotide.
 2. The method of claim 1, further comprising the step of treating said annealed oligonucleotides with a ligating enzyme.
 3. The method of claim 1, further comprising the step of amplifying said double-stranded polynucleotide.
 4. The method of claim 1, wherein said double-stranded polynucleotide comprises a length selected from the group consisting of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10×10³, 20×10³, 30×10³, 40×10³, 50×10³, 60×10³, 70×10³, 80×10³, 90×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹ and 1×10¹⁰ base pairs.
 5. The method of claim 1, wherein said double-stranded polynucleotide comprises a coding region and a corresponding regulatory element directing the expression of said coding region.
 6. The method of claim 5, wherein said regulatory element further comprises a promoter.
 7. The method of claim 1, wherein said double-stranded polynucleotide further comprises a plurality of coding regions and a plurality of regulatory elements.
 8. The method of claim 7, wherein said coding regions encode products that comprise a biochemical pathway.
 9. The method of claim 8, wherein said biochemical pathway is glycolysis.
 10. The method of claim 9, wherein said coding regions encode enzymes selected from the group consisting of hexokinase, phosphohexose isomerase, phosphofructokinase-1, aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase.
 11. The method of claim 8, wherein said biochemical pathway is lipid synthesis.
 12. The method claim 7, wherein said biochemical pathway is cofactor synthesis.
 13. The method of claim 7, wherein said pathway involves lipoic acid.
 14. The method of claim 7, wherein said biochemical pathway is riboflavin synthesis.
 15. The method of claim 7, wherein said biochemical pathway is nucleotide synthesis.
 16. The method of claim 15, wherein said nucleotide is a purine.
 17. The method of claim 15, wherein said nucleotide is a pyrimidine.
 18. The method of claim 7, wherein said coding regions encode enzymes involved in a cellular process selected from the group consisting of cell division, chaperone, detoxification, peptide secretion, energy metabolism, regulatory function, DNA replication, transcription, RNA processing and tRNA modification.
 19. The method of claim 18, wherein said energy metabolism is oxidative phosphorylation.
 20. The method of claim 1, wherein said double-stranded polynucleotide is a DNA.
 21. The method of claim 1, wherein said double-stranded polynucleotide is an RNA.
 22. The method of claim 1, wherein said double-stranded polynucleotide is an expression construct.
 23. The method of claim 22, wherein said expression construct is a bacterial expression construct.
 24. The method of claim 22, wherein said expression construct is a mammalian expression construct.
 25. The method of claim 17, wherein said expression construct is a viral expression construct.
 26. The method of claim 1, wherein said double-stranded polynucleotide comprises a genome selected from the group consisting of bacterial genome, yeast genome, viral genome, mammalian genome, amphibian genome and avian genome.
 27. The method of claim 1, wherein each said overlap is between about 5 base pairs and about 75 base pairs.
 28. The method of claim 1, wherein said overlap is selected form the group consisting of about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 30 base pairs, about 35 base pairs, about base pairs, about 45 base pairs, about 50 base pairs, about 55 base pairs, about 60 base pairs, about 65 base pairs, and about 70 base pairs.
 29. The method of claim 5, wherein said promoter is selected from the group consisting of CMV IE, SV40 IE, RSV, β-actin, tetracycline regulatable and ecdysone regulatable.
 30. The method of claim 26, wherein said genome is a viral genome.
 31. The method of claim 30, wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.
 32. The method of claim 1, wherein said double-stranded polynucleotide is a chromosome.
 33. A method of producing a viral particle comprising the steps of: (a) providing a host cell; (b) transforming said host cell with an artificial viral genome prepared by: (i) generating a first set of oligonucleotides comprising one strand of said viral genome; (ii) generating a second set of oligonucleotides comprising the second complementary strand of said viral genome; and (iii) annealing said first and said second set of oligonucleotides; wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of said viral genome will hybridize with only one complementary oligonucleotide; and (c) culturing said transformed host cell under conditions such that said viral particle is expressed.
 34. The method of claim 33, wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.
 35. The method of claim 1, wherein said double-stranded polynucleotide comprises an artificial chromosome, wherein said chromosome comprises all coding regions and regulatory elements found in a corresponding natural chromosome.
 36. The method of claim 35, wherein said corresponding natural chromosome is a human mitochondrial genome.
 37. The method of claim 35, wherein said corresponding natural chromosome is a chloroplast genome.
 38. The method of claim 1, wherein said double-stranded polynucleotide comprises an artificial genetic system, wherein said system comprises all coding regions and regulatory elements found in a corresponding natural biochemical pathway.
 39. The method of claim 38, wherein said biochemical pathway comprises the activities required for glycolysis.
 40. The method of claim 38, wherein said biochemical pathway comprises the enzymes required for electron transport.
 41. The method of claim 38, wherein said biochemical pathway comprises the enzyme activities required for photosynthesis.
 42. The method of claim 1, wherein said next most terminal oligonucleotide of said double-stranded polynucleotide has an overlap of about 50 percent with said first annealed product.
 43. A method of synthesizing a replication-competent double-stranded polynucleotide, comprising: (a) annealing a 5′ terminal oligonucleotide with a 3′ terminal oliogonucleotide of a replication-competent double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal oligonucleotide of said replication-competent double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, and (b) repeating step (a) one or more times to sequentially produce a replication-competent double-stranded polynucleotide.
 44. A method of synthesizing a double-stranded polynucleotide, comprising: (a) annealing a 5′ terminal oligonucleotide with a 3′ terminal oliogonucleotide of a double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, said next most terminal double-stranded polynucleotide comprising at least one oligonucleotide of at least about 25 bases, and (b) repeating step (a) one or more times to sequentially produce a double-stranded polynucleotide.
 45. A method of synthesizing a replication-competent double-stranded polynucleotide, comprising: (a) annealing a 5′ terminal oligonucleotide with a 3′ terminal oliogonucleotide of a replication-competent double-stranded polynucleotide to produce a first annealed product and directly adding a next most terminal double-stranded polynucleotide to said first annealed product under conditions sufficient for annealing to produce a second annealed product, and (b) repeating step (a) one or more times to sequentially produce a replication-competent double-stranded polynucleotide. 